ProjectSummary Thisproposedworkseekstodevelopasuiteofenablingtechnologiescapableofproducingsynthetic phosphoproteinswiththegoaloftransformingthefieldofhumanproteinsignalingfromonethatispurely observation?basedintoonethatbiosynthesizesdesignerproteinstoachieveacomprehensiveunderstandingof complexsignalingnetworks.Theimportanceofphosphorylationisemphasizedbythefactthat phosphorylatedproteinscontrolmostaspectsofnormalcellularhomeostasis.Aberrationsinprotein phosphorylationcandrivecancer,hypertension,diabetes,andneurodegenerativedisorders.Thus, understandingdifferentialpatternsofproteinphosphorylationindiseasestatesisofextremephysiological andclinicalinterest.Analysisofphosphorylatedaminoacidresidueshasbeenlimitedbyourinabilityto controlthesechemicalmodificationsduetoalackofphosphomimeticsthatfullyrecapitulatethechemistryof phosphorylatedresidues.Currentprogresstowardtheelucidationofphospho?signalingnetworksis hamperedbythelackofmethodstoproduceproteinscontainingspecificcombinationsofphosphorylated aminoacids.Inparticular,syntheticchemistryisinadequatefortotalphosphoproteinsynthesis,and conventionalbiologicalmethodsdonotcontrolphosphorylationlevels.Wehaverecentlydevelopedanew technology,albeitlimitedtophosphoserine(pSer),thatenablesthesynthesisofrecombinantphosphoproteins. Thistechnologydirectsphosphorylatedaminoacidsintotheirphysiologicallyrelevantpositionswithin proteinsyetourfunctionalunderstandingofproteinphosphorylationwillremainincompletewithoutaccess tophosphotyrosine(pTyr)andphosphothreonine(pThr)containingproteins.SpecificAims:InAim1,wewill utilizemutagenesisandlaboratoryevolutiontoengineeranoptimizedtyrosylaminoacyl?tRNAsynthetasefor phosphotyrosine.InAim2,wewillprovideasolutiontothisproblembyengineeringanaminoacyl?tRNA synthetasethatcanchargeaphosphothreonineontoaspecialtRNAthatreadsadedicatedopencodon. Uniquetoourapproach,wewillalsoemployourgenomicallyrecodedE.colicellsinwhichopenstopcodons canbeconvertedintonewsensecodonsthatencodepThrandpTyrintopreciselocationsinrecombinant proteins.Significance:Theoveralloutcomeofourstudieswillbeanenablingtechnologyfortheexpressionof pTyrandpThrcontainingproteinsthatwillbroadlyenableresearchintodiseasemechanismsandcanbeused directlytodevelopnewtherapiesforhumandisease.Thiswillbethefirsttechnologyabletore?createhuman diseasenetworksthatare?difficult?or?impossible?toinfiltrate,andwillestablishtheparadigmfor addressingotherpost?translationalmodifications.Morebroadly,theproposedworkwillenablethere?design ofprogrammablesignalingnetworkscomprisingproteinswithnaturalandsyntheticnonstandardaminoacids capableofexpandingnetworksbeyondtheirnaturalfunctionsandofproducingnovelsyntheticpolymers withdiversechemistries.